The long term goal of this grant is to determine how proteins that interact with yeast telomeres affect telomere function. In most eukaryotes, including Saccharomyces cerevisiae, telomeric DNA is synthesized by a specialized reverse transcriptase, telomerase, in yeast, tetomerase action is both cell cycle regulated, occurring in late S phase, and length regulated, occurring preferentially at short telomeres. Core telomerase consists of telomerase RNA, encoded by the TLC1 gene, and the catalytic reverse transcriptase, encoded by EST2. Cdcl3p, which binds the single strand TGI__ tails at the very ends of yeast chromosomes is also essential for tetomerase action in vivo as are Estlp and Est3p, two proteins whose functions are poorly understood. In addition, Teltp, an ATM-like kinase and the Mrel 1-Rad50-Xrs2 (MRX) complex function in the same pathway to promote telomerase action in vivo. In the last funding period, we developed a sensitive chromatin immuno-precipitation assay that can detect Est2p, Estlp, and Cdc 13p at telomeres. This assay reveals that Cdcl3p is tetomere bound throughout the cell cycle but its binding increases greatly in late S phase, the time of telomerase action, Est2p binds telomeres not just in late S phase when telomerase is acti',,e but also in G1 and early S phase when it is not. Estlp binding is restricted to late S phase. These data suggest a model where Cdc 13p multimerization and Estt p binding activate an otherwise inactive, telomere bound Est2plTLC t complex. This grant proposes experiments to test and extend this model, The role of Est lp will be further defined, using both in vivo and in vitro assays. We wilt determine if the Ku complex holds Est2p/TLCt at the telomere in Gt and if its presence is needed for the capping function of telomeres. We will determine if Est3p and MRX bind telomeres, if this binding is important for their role in promoting telomerase, and if their absence affects binding of other tetomerase components. We will determine if the preferential lengthening of short telomeres is Estlp and/or MRX mediated. The last aim describes cytological approaches to determine if telornere localization to the nuclear periphery is important for transcriptional silencing or for telomerase lengthening of telomeres. There is increasing evidence that telomere replication has effects on both aging and cancer. Telomere functions, including telomeric silencing, as well as the proteins that act at tetomeres are conserved from yeasts to humans. Analysis of proteins that affect yeast telomeres is likely to be relevant to an understanding of genetic instability in humans. city, state)